Structure-Activity Based Mechanistic Insights into Cleavage Chemistry by Self-Cleaving Nucleolytic Ribozymes

基于结构-活性的自裂解核酶裂解化学的机理见解

• 批准号: 10684151
• 负责人: DINSHAW J PATEL
• 金额: $ 33.63万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684151
• 关键词: 3-Dimensional; Acids; Active Sites; Address; Architecture; Binding; Binding Sites; Biochemical; Biogenesis; Biological; Catalysis; Catalytic RNA; Cations; Chemicals; Chemistry; Collaborations; Conserved Sequence; Crystallization; Curiosities; Dependence; Divalent Cations; Elements; Enzymes; Event; Evolution; Formulation; Gene Expression Regulation; Genes; Genomics; Goals; Hepatitis Delta Virus; Human; Hydration status; Hydrogen Bonding; Hydrophobic Interactions; Introns; Life; Ligation; Literature; Measurement; Measures; Mediating; Messenger RNA; Modification; Molecular Conformation; Monitor; Oxygen; Peptides; Peptidyltransferase; Play; Positioning Attribute; Publishing; RNA; RNA Processing; RNA Splicing; RNase P; Reporting; Ribosomes; Role; Screening Result; Sister; Site; Specificity; Spliceosomes; Structure; Structure-Activity Relationship; Temperature; Time; Untranslated RNA; Untranslated Regions; Vanadates; Vertebral column; Visit; analog; base; comparative; follow-up; functional group; genome integrity; genome wide screen; inorganic phosphate; insight; interest; macromolecule; mutant; phosphodiester; prebiotics; protonation; varkud satellite ribozyme

ABSTRACT Our mechanistic understanding of small (<150 nt) self-cleaving nucleolytic ribozymes that primarily use general- acid base catalysis involving attack of the 2’-OH on the adjacent scissile phosphate to site-specifically cleave the intervening phosphodiester backbone has initially focused on hammerhead, hairpin, glmS, hepatitis delta virus and Varkud Satellite ribozymes, and more recently on twister (Twr), twister-sister (TSr), pistol (Psr) and hatchet (Htr) ribozymes. Our overall goal as reflected from our structure-function studies of the Twr, Tsr, Psr and Htr ribozymes is to expand on our current understanding of the catalytic versatility of RNA with the emphasis on the contributions of active site organization, geometric constraints, activation of the 2’-OH nucleophile, the role of transition state stabilization, protonation of the 5’-oxygen leaving group and the potential of Mg2+ cations in mediating catalysis. One of the challenges in the field relates to whether self-cleaving ribozymes use a common or diverse set of mechanisms, and the extent to which hydrated divalent cations catalyze cleavage chemistry. We have an ongoing collaboration with the Ronald Micura lab (Innsbruck) to study catalytic mechanisms of self- cleaving ribozymes by solving crystal structures of precatalytic, transition and product states in our lab, followed by systematic multi-faceted studies of structure-guided selective catalytic mutants and analogs, as well as pKa measurements of catalytic residues, and pH and temperature-dependence of catalytic rates by the Micura lab. Aim 1: A recent biochemical genome-wide screen resulted in the identification of the naturally occurring self- cleaving hovlinc ribozyme in humans. The sequence of the 168-nt hovlinc ribozyme and its 83-nt minimal functional counterpart contained two pseudoknots with one of them embedded in the cleavage site. The cleavage rate was shown to increase with pH and its inverse correlation with the pKa of divalent cations suggested the catalytic participation of a hydrated divalent cation in cleavage chemistry. We propose to crystallize and determine the structures of the precatalytic, vanadate transition-state mimic and product states of the minimal functional hovlinc ribozyme and follow up with systematic functional studies with the Micura lab of structure- guided modifications and rate measurements towards elucidation of its catalytic cleavage mechanism. Aim 2. This Aim revisits structure-activity relationships of the Twr and TSr ribozymes to resolve discrepancies in the published precatalytic structures and resulting mechanistic insights reported in the literature. The splayed- apart orientation of bases at the cleavage site in a four-way junctional TSr ribozyme by our group contrasts with the stacked bases at the cleavage site in a three-way junctional TSr from the David Lilley lab. We propose to characterize vanadate transition-state mimics of the Twr and TSr ribozymes to resolve the existing discrepancies and mechanistically evaluate the proposed role of catalytic bases and hydrated divalent cations in mediating cleavage chemistry. These studies should elucidate whether Twr and TSr ribozymes, that contain common but topologically distinct conserved sequence elements, use similar or distinct mechanisms for cleavage chemistry.

抽象的 我们对小（<150 nt）自裂解的核酸核酶的机械理解，这些核酶主要用途 - 酸碱催化涉及2'-OH对相邻cissile磷酸盐的攻击至特定于位点的清除 中间的磷酸化骨架最初集中于锤头，发夹，GLM，肝炎三角洲 病毒和varkud卫星核酶，以及最近在Twister（TWR），Twister-Sister（TSR），手枪（PSR）和 斧头（HTR）核酶。我们对TWR，TSR，PSR和 HTR核酶是扩展我们当前对RNA催化多功能性的理解，重点是 主动站点组织，几何约束，2'-OH核pholipile的激活的贡献，该角色 过渡状态稳定，5'-氧的质子化离开组的质子化以及Mg2+阳离子的潜力 介导催化。该领域的挑战之一是关于自我切换的核酶是否使用常见 或各种机制，以及水合分裂的阳离子在催化裂解化学的程度。 我们与罗纳德·米库拉（Ronald Mikura）实验室（因斯布鲁克（Innsbruck））进行了持续的合作，以研究自我的催化机制 通过在我们实验室中求解预催化，过渡和产物态的晶体结构来切割核酶，然后 通过系统的多面研究结构引导的选择性催化突变体和类似物以及PKA Mikura Lab测量催化保留，pH和催化速率的温度依赖性。 AIM 1：最近的生化基因组筛选导致鉴定出天然发生的自我 在人类中裂解hovlinc核酶。 168-nt Hovlinc核酶的序列及其83 nt最小 功能对应物包含两个伪诺，其中一个嵌入了裂解位点。乳沟 证明速率随pH的增加及其与二价阳离子的PKA的逆相关性表明 水合二价阳离子在切割化学中的催化参与。我们建议结晶和 确定催化前的，钒酸盐过渡状态的模拟和最小值的产物状态 功能性霍夫林核酶，并通过结构的Mikura实验室进行系统的功能研究 指导的修改和速率测量值阐明了其催化裂解机制。 目标2。此目标重新审视TWR和TSR核酶的结构活性关系以解决差异 在文献中报道的已发表的预先分析结构和产生的机械见解中。展示的 在我们的组中，在四向连接的TSR核酶中裂解位点的碱取向与我们的组对比 David Lilley Lab的三向连接式TSR中的裂解位置堆叠的基地。我们建议 表征TWR和TSR核酶的杂物过渡状态模仿以解决现有差异 并机械评估催化碱和水合二价阳离子中提出的作用 切割化学。这些研究应阐明是否包含常见但 拓扑上不同的保守序列元素，使用相似或不同的机制进行切割化学。